Glycosylation of anthocyanin plays an important role in increasing its stability and diversity in plants. Here, we identified a glucosyltransferase gene responsible for the anthocyanin components in Litchi chinensis Sonn. Cyanidin-3-rutinoside is the main anthocyanins of pericarps and red hairy roots overexpressing MYB transcription factor LcMYB1 in the late maturing cultivars. However, in the early maturing cultivars, the anthocyanins in the pericarps is dominated by cyanidin-3-glucoside, and the anthocyanins in the red hairy roots overexpressing LcMYB1 is dominated by cyanidin-3-rutinoside. Enzyme assays highlighted notable differences in flavonoid 3-O-rhamnosyltransferase (F3RT) activity between the pericarps and red hairy roots overexpressing LcMYB1 in the early maturing cultivars. Two differentially expressed genes (DEGs), LcF3RT1 and LcF3RT2, were significantly up-regulated in the red hairy roots overexpressing LcMYB1. Yeast one-hybrid and dual luciferase reporter assays revealed that LcMYB1 could bind to the promoter of LcF3RT2 and significantly activate its expression. Functional validation showed that LcF3RT2 could catalyze the conversion of cyanidin-3-glucoside into cyanidin-3-rutinoside, leading to the differences on anthocyanin components in pericarps and red hairy roots of early maturing litchi cultivars. Our results will provide insights into the regulation and glycosylation modification of anthocyanins in litchi as well as in other plants.